The Essentiality of DivIVAEf Oligomerization for Proper Cell Division in Enterococcus faecalis and Interaction with a Novel Cell Division Protein
Previous detection of DivIVA interaction with a novel cell division protein, MLJD1, by screening a Yeast Two-Hybrid (Y2H) was weak. GST-pulldown and immunoprecipitation did indicate DivIVAEf interaction with MLJD1, but another in vivo assay was required to support these results. In this study I demonstrate a strong interaction, using an in vivo Bacterial Two-Hybrid (B2H) assay, between DivIVAEf and a fragment of MLJD1 containing two cystathionine-beta-synthase (CBS) domains. The in vitro and in vivo results thus confirm interaction between DivIVAEf and MLJD1.
Another objective of this study was to determine the localization of DivIVA and MLJD1 in E. faecalis. Localization of DivIVAEf in E. faecalis was found to be similar to DivIVA localization in Bacillus subtilis and Streptococcus pneumonia. DivIVAEf was diffused along the cell membrane and, as chromosome replication and segregation and cell division proceeded, DivIVAEf migrated to the cell poles and then concurrently to the division site. Intriguingly, MLJD1 was found to localize in the same pattern as DivIVAEf in E. faecalis, further implicating MLJD1 as a bacterial cell division protein.
Since MLJD1 has potential DNA binding capabilities a proposed model of its role in cell division has been proposed. I hypothesize that MLJD1 could be forming a bridge between DivIVAEf and the chromosome to aid in proper chromosomal replication and segregation. This model could explain how DivIVAEf is involved in chromosome replication. This model is similar to the role of RacA in sporulation in B. subtilis where RacA directs the chromosome during sporulation through direct interaction with DivIVABs and Spo0J.
This study has set some important and essential ground work for developing a novel model of cell division for the elusive Gram-positive coccal bacterial strains.
Advisor:Dr. George G. Khachatourians; Dr. Jo-Anne R. Dillon; Dr. Peter Howard; Dr. Sean Hemmingsen; Dr. Vikram Misra
School:University of Saskatchewan
School Location:Canada - Saskatchewan
Source Type:Master's Thesis
Keywords:i enterococcus faecalis protein interactions cell division diviva oligomerization immunofluorescence microscopy bacterial two hybrid
Date of Publication:04/15/2009